Metalloinsertor complexes targeted to dna mismatches

ABSTRACT

A composition including a Rh or Ru metalloinsertor complex specifically targets mismatch repair (MMR)-deficient cells. Selective cytotoxicity is induced in MMR-deficient cells upon uptake of the inventive metalloinsertor complexes.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application claims priority to and the benefit of U.S.Provisional Application Ser. No. 61/484,514 filed on May 10, 2011, andU.S. Provisional Application Ser. No. 61/613,292 filed on Mar. 20, 2012,the entire contents of both of which are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM033309 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD

This disclosure is directed to metalloinsertor complexes and the use ofthese complexes to selectively target specific deficient cells.

BACKGROUND

Mismatches in DNA arise naturally as a result of replication errors,endogenous DNA damaging agents, and spontaneous processes such ascytosine deamination. The mismatch repair (MMR) pathway acts to correctmismatches before subsequent rounds of replication, reducing the numberof DNA mismatches in the human genome from ˜1000 to ˜1. The loss of MMRcarries dire consequences, including increased mutation rates,carcinogenesis, and resistance to a variety of clinical anti-canceragents, such as anti-metabolites, DNA alkylators, and cisplatin. Indeed,deficiencies in MMR have been linked to a variety of cancers, inparticular, nonhereditary colorectal carcinoma, and are also associatedwith resistance or tolerance to many common therapeutics. Furthermore,this resistance to commonly used agents leads to enrichment ofMMR-deficient cells; roughly half of secondary leukemias showMMR-deficiency. These issues point to the need for a therapeutic agentthat specifically targets MMR-deficient cells. Of course, any potentialagent must first reach its target before it may bind.

To that end, metalloinsertors have been developed to target DNAmismatches in vitro. DNA mismatches, owing to their loss of hydrogenbonding and poor stacking, are destabilized relative to well matchedDNA. It is this thermodynamic destabilization that allows for a means oftargeting mismatches, since mismatches do not significantly perturb thestructure of the B-form DNA duplex. However, while existingmetalloinsertors have been used to detect the existence of DNAmismatches, to date, the existing metalloinsertors have not been shownto cause cell death.

SUMMARY

Embodiments of the present invention are directed to a metalloinsertorcomplex capable of specifically targeting MMR-deficient cells.

In some embodiments of the present invention, a composition includes acomplex represented by Formula I, depicted below.

M^(m+)(L₁)(L₂)(L₃)(L₄)(L₅)  Formula I

In Formula I, M is rhodium or ruthenium, and m is 2 or 3. L₁ isbenzo[a]phenazine-5,6-diimine (phzi) or chrysene-5,6-diimine (chrysi),as depicted below.

In Formula I, each of L₂ through L₅ is either NH₃ or combines with anadjacent one of L₂ through L₅ to form a single ligand with twocoordination sites to M, where the single ligand with two coordinationsites to M is selected from:

orIn Formula II and Formula III, R₁ is selected from alkyl groupsterminating in one selected from CH₃, OH, SH, NH₂, COOH, N₃, oralkynyl-linked peptide moieties, and PEGylated groups terminating in oneselected from CH₃, OH, SH, NH₂, COOH, N₃, or alkynyl-linked peptidemoieties.

In some embodiments of the present invention, a method for selectivelyinducing cytotoxicity in mismatch repair (MMR)-deficient cells includesproviding a metalloinsertor complex of Formula I to the MMR-deficientcells. Here, a decrease in cell viability in the MMR-deficient cellsthat does not, or would not comparably decrease cell viability inMMR-proficient cells, indicates selective cytotoxicity in MMR-deficientcells.

In some embodiments, a method of decreasing MMR-deficient cellproliferation includes providing a metalloinsertor complex of Formula Ito MMR-deficient cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the structure of the rhodium metalloinsertor[Rh(DPK)(NH₃)₂chrysi]³⁺, according to embodiments of the presentinvention.

FIG. 1B is the structure of the rhodium metalloinsertor[Rh(DPE)(NH₃)₂chrysi]³⁺, according to embodiments of the presentinvention.

FIG. 1C is the structure of the rhodium metalloinsertor[Rh(NH₃)₄phzi]³⁺, according to embodiments of the present invention.

FIG. 1D is the structure of the rhodium metalloinsertor[Rh(HDPA)₂phzi]³⁺, according to embodiments of the present invention.

FIG. 1E is the structure of the rhodium metalloinsertor[Rh(MeDPA)₂phzi]³⁺, according to embodiments of the present invention.

FIG. 1F is the structure of the rhodium metalloinsertor[Rh(MeDPA)₂chrysi]³⁺, according to embodiments of the present invention.

FIG. 1G is the structure of the rhodium metalloinsertor[Rh(MDPA)₂(phen)(chrysi)]³⁺, according to embodiments of the presentinvention.

FIG. 1H is the structure of the rhodium metalloinsertor[Rh(PrDPA)₂(phen)(chrysi)]³⁺, according to embodiments of the presentinvention.

FIG. 1J is the structure of the rhodium metalloinsertor[Rh(HexDPA)₂(phen)(chrysi)]³⁺, according to embodiments of the presentinvention.

FIG. 1K is the structure of the rhodium metalloinsertor[Rh(PrDPA)₂chrysi]³⁺, according to embodiments of the present invention.

FIG. 1L is the structure of the rhodium metalloinsertor[Rh(DPAE)₂chrysi]³⁺, according to embodiments of the present invention.

FIG. 1M is the structure of the rhodium metalloinsertor[Rh(HDPA)(phen)(chrysi)]³⁺, according to embodiments of the presentinvention.

FIG. 1N is the structure of the rhodium metalloinsertor[Rh(DPE)(phen)(chrysi)]³⁺, according to embodiments of the presentinvention.

FIG. 2 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(DPK)(NH₃)₂chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 24 hour incubation, accordingto embodiments of the present invention.

FIG. 3 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(DPE)(NH₃)₂chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 4 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(NH₃)₄phzi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 24 hour incubation, according toembodiments of the present invention.

FIG. 5 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(HDPA)₂phzi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 24 hour incubation, according toembodiments of the present invention.

FIG. 6 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(DPE)(NH₃)₂phzi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 24 hour incubation, according toembodiments of the present invention.

FIG. 7 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(MeDPA)2chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 24 hour incubation, according toembodiments of the present invention.

FIG. 8 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(phen)(MeDPA)chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 9 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(phen)(EtDPA)chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 10 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(phen)(PrDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 11 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(phen)(hexylDPA)chrysi]³⁺ concentration in the HCT116N (squares)and the HCT116O (circles) cell lines after a 72 hour incubation,according to embodiments of the present invention.

FIG. 12 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(phen)(HDPA)chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 13 is a graph of the amount (%) of BrdU incorporation as a functionof [Rh(phen)(DPE)chrysi]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 24 hour incubation, accordingto embodiments of the present invention.

FIG. 14A is a graph of the amount (%) of BrdU incorporation as afunction of [Rh(PropylDPA)₂chrysi]³⁺ concentration in the HCT116N(squares) and the HCT116O (circles) cell lines after a 24 hourincubation, according to embodiments of the present invention.

FIG. 14B is a graph of the amount (%) of BrdU incorporation as afunction of [Rh(PropylDPA)₂chrysi]³⁺ concentration in the HCT116N(squares) and the HCT116O (circles) cell lines after a 72 hourincubation, according to embodiments of the present invention.

FIG. 15A is a graph of the amount (%) of BrdU incorporation as afunction of [Rh(DPAE)₂chrysi]³⁺ concentration in the HCT116N (squares)and the HCT116O (circles) cell lines after a 6 hour incubation,according to embodiments of the present invention.

FIG. 15B is a graph of the amount (%) of BrdU incorporation as afunction of [Rh(DPAE)₂chrysi]³⁺ concentration in the HCT116N (squares)and the HCT116O (circles) cell lines after a 24 hour incubation,according to embodiments of the present invention.

FIG. 16 is a graph showing the percent of differential inhibition ofcellular proliferation between the HCT116N and the HCT116O cells whenincubated with 10 μM of the rhodium complexes as indicated, for 24hours, followed by BrdU incorporation and quantification, according toembodiments of the present invention.

FIG. 17A is a graph of the amount (%) of viable cells as a function of[Rh(DPAE)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 24 hour incubation, according toembodiments of the present invention.

FIG. 17B is a graph of the amount (%) of viable cells as a function of[Rh(DPAE)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 48 hour incubation, according toembodiments of the present invention.

FIG. 17C is a graph of the amount (%) of viable cells as a function of[Rh(DPAE)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 18A is a graph of the amount (%) of viable cells as a function of[Rh(PrDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cells after a 24 hour incubation, according toembodiments of the present invention.

FIG. 18B is a graph of the amount (%) of viable cells as a function of[Rh(PrDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 48 hour incubation, according toembodiments of the present invention.

FIG. 18C is a graph of the amount (%) of viable cells as a function of[Rh(PrDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 19A is a graph of the amount (%) of viable cells as a function of[Rh(bpy)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 48 hour incubation, according toembodiments of the present invention.

FIG. 19B is a graph of the amount (%) of viable cells as a function of[Rh(bpy)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 20A is a graph of the amount (%) of viable cells as a function of[Rh(HDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 48 hour incubation, according toembodiments of the present invention.

FIG. 20B is a graph of the amount (%) of viable cells as a function of[Rh(HDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 21A is a graph of the amount (%) of viable cells as a function of[Rh(MeDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 48 hour incubation, according toembodiments of the present invention.

FIG. 21B is a graph of the amount (%) of viable cells as a function of[Rh(MeDPA)₂chrysi]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 22 is a graph of the amount (%) of viable cells as a function of[Rh(NH₃)₄phzi]³⁺ concentration in the HCT116N (squares) and the HCT116O(circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 23 is a graph of the amount (%) of viable cells as a function of[Rh(chrysi)(phen)(DPE)]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 24 is a graph of the amount (%) of viable cells as a function of[Rh(chrysi)(phen)(HDPA)]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 25 is a graph of the amount (%) of viable cells as a function of[Rh(chrysi)(phen)(MeDPA)]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 26 is a graph of the amount (%) of viable cells as a function of[Rh(chrysi)(phen)(PrDPA)]³⁺ concentration in the HCT116N (squares) andthe HCT116O (circles) cell lines after a 72 hour incubation, accordingto embodiments of the present invention.

FIG. 27 is a graph of the amount (%) of viable cells as a function of[Rh(DIP)₂(chrysi)]³⁺ concentration in the HCT116N (squares) and theHCT116O (circles) cell lines after a 72 hour incubation, according toembodiments of the present invention.

FIG. 28 is a schematic of the synthesis of DPAE (5a) and PrDPA (5b) fromdipyridylamine (2), according to embodiments of the present invention.

FIG. 29 is a schematic of the synthesis of rac-[Rh(DPAE)₂(chrysi)]³⁺(1a) and rac-[Rh(PrDPA)₂(chrysi)]³⁺(1b), according to embodiments of thepresent invention.

FIG. 30A is an autoradiogram of a 20% denaturing polyacrylamide gel of³²P-labeled hairpin DNA in the presence and absence ofrac-[Rh(DPAE)₂(chrysi)]³⁺.

FIG. 30B is an autoradiogram of a 20% denaturing polyacrylamide gel of³²P-labeled hairpin DNA in the presence and absence ofrac-[Rh(PrDPA)₂(chrysi)]³⁺.

FIG. 30C is a sigmoidal curve fits graph of binding affinity competitiontitrations calculated from the autoradiograms of FIG. 30A for[Rh(DPAE)₂(chrysi)]³⁺ (squares) and of FIG. 30B for[Rh(PrDPA)₂(chrysi)]³⁺ (circles), according to embodiments of thepresent invention.

FIG. 31A is a graph of the results from an ICP-MS (inductively coupledplasma mass spectrometry) assay quantifying the uptake of[Rh(DPAE)₂(chrysi)]³⁺ (“DPAE”) and [Rh(PrDPA)₂(chrysi)]³⁺ (“PrDPA”) inwhole cell extracts of HCT116O cells, according to embodiments of thepresent invention.

FIG. 31B is a graph of the results from an ICP-MS assay quantifying theuptake of [Rh(DPAE)₂(chrysi)]³⁺ (“DPAE”)(solid black) and[Rh(PrDPA)4chrysi)]³⁺ (“PrDPA”) (hashed lines) in nuclear andmitochondrial fractions of HCT116O cells, according to embodiments ofthe present invention.

FIG. 32 is a graph of the results from an ICP-MS assay for rhodiumaccumulation in whole cell lysates of HCT116N (solid with lines) andHCT116O (solid) cells treated with No Rh, or 10 μm of[Rh(bpy)₂chrysi]³⁺, [Rh(HDPA)₂chrysi]³⁺, or [Rh(NH₃)₄chrysi]³⁺ (ammine)for 48 hours, according to embodiments of the present invention.

FIG. 33A is a graph of the results from an ICP-MS assay for quantifyingthe whole-cell rhodium accumulation in HCT116O cells treated with 10 μmof each of the indicated rhodium complexes, or 2 μm of[Rh(DIP)₂(chrysi)]³⁺ complex for 1, 3, 6, 12, or 24 hours (left toright), according to embodiments of the present invention.

FIG. 33B is a graph of the results from an ICP-MS assay for quantifyingthe rhodium accumulation in the nucleus (right y-axis, right bars) andmitochondria (left y-axis, left bars) of HCT116O cells treated with 10μm of each of the indicated rhodium complexes, or 2 μm of[Rh(DIP)₂(chrysi)]³⁺ complex for 24 hours, according to embodiments ofthe present invention.

FIG. 34A is a graph comparing the cell cycle distributions of untreatedHCT116N cells (dark gray) and HCT116N cells after treatment with 20 μm[Rh(HDPA)₂chrysi]³⁺ (light gray) for a 24 hour incubation, followed byfixation and staining with propidium iodide (PI) and flow cytometryanalysis, according to embodiments of the present invention.

FIG. 34B is a graph comparing the cell cycle distributions of untreatedHCT116N cells (dark gray) and HCT116N cells after treatment with 20 μm[Rh(HDPA)₂chrysi]³⁺ (light gray) for a 48 hour incubation, followed byfixation and staining with propidium iodide (PI) and flow cytometryanalysis, according to embodiments of the present invention.

FIG. 34C is a graph comparing the cell cycle distributions of untreatedHCT116O cells (dark gray) and HCT116O cells after treatment with 20 μm[Rh(HDPA)₂chrysi]³⁺ (light gray) for a 24 hour incubation, followed byfixation and staining with propidium iodide (PI) and flow cytometryanalysis, according to embodiments of the present invention.

FIG. 34D is a graph comparing the cell cycle distributions of untreatedHCT116O cells (dark gray) and HCT116O cells after treatment with 20 μm[Rh(HDPA)₂chrysi]³⁺ (light gray) for a 48 hour incubation, followed byfixation and staining with propidium iodide (PI) and flow cytometryanalysis, according to embodiments of the present invention.

FIG. 35A is a graph of the cell cycle phase distribution (G (far left),S (middle), and G2/M (far right)) in the untreated HCT116N cells (−) andthe [Rh(HDPA)₂chrysi]³⁺ treated HCT116N cells (+) from FIGS. 34A and34B, according to embodiments of the present invention.

FIG. 35B is a graph of the cell cycle phase distribution (G (far left),S (middle), and G2/M (far right)) in the untreated HCT116O cells (−) andthe [Rh(HDPA)₂chrysi]³⁺ treated HCT116O cells (+) from FIGS. 34C and34D, according to embodiments of the present invention.

FIG. 36A is a distribution plotting the fluorescence from a populationof cells stained with YO-PRO-1 and PI in HCT116N cells, according toembodiments of the present invention.

FIG. 36B is a distribution plotting the fluorescence from a populationof cells stained with YO-PRO-1 and PI in HCT116N cells after treatmentwith 20 μM [Rh(HDPA)₂ chrysi]³⁺ for a 72 hour incubation, according toembodiments of the present invention.

FIG. 36C is a distribution plotting the fluorescence from a populationof cells stained with YO-PRO-1 and PI in HCT116O cells, according toembodiments of the present invention.

FIG. 36D is a distribution plotting the fluorescence from a populationof cells stained with YO-PRO-1 and PI in HCT116O cells after treatmentwith 20 μM [Rh(HDPA)₂ chrysi]³⁺ for a 72 hour incubation, according toembodiments of the present invention.

FIG. 37A is a graph of the number of cells that are Live, Apoptotic,Necrotic, and Dead determined from the raw fluorescence data of theHCT116N cells of FIGS. 36A and 36B, according to embodiments of thepresent invention.

FIG. 37B is a graph of the number of cells that are Live, Apoptotic,Necrotic, and Dead determined from the raw fluorescence data of theHCT116O cells of FIGS. 36C and 36D, according to embodiments of thepresent invention.

FIG. 38A is a graph comparing the percent viability of HCT116N (solid)and HCT116O (lined) cells treated with 0, 15, and 30 μm[Rh(HDPA)₂chrysi]³⁺ with or without 20 μm of the pan-caspase inhibitorZ-VAD-FMK for a 24 hour incubation, and labeled with MTT, according toembodiments of the present invention.

FIG. 38B is a graph comparing the percent viability of HCT116N andHCT116O cells treated with 0, 15, and 30 μm [Rh(HDPA)₂chrysi]³⁺ with orwithout 20 μm of the pan-caspase inhibitor Z-VAD-FMK for a 48 hourincubation, and labeled with MTT, according to embodiments of thepresent invention.

FIG. 38C is a graph comparing the percent viability of HCT116N andHCT116O cells treated with 0, 15, and 30 μm [Rh(HDPA)2chrysi]³⁺ with orwithout 20 μm of the pan-caspase inhibitor Z-VAD-FMK for a 72 hourincubation, and labeled with MTT, according to embodiments of thepresent invention.

FIG. 39A is a graph comparing the percent viability of HCT116N (solid)and HCT116O (lined) cells treated with (+=25 μM, or ++=50 μM) or withoutthe PARP inhibitor DPQ, and with or without 20 μM [Rh(HDPA)₂chrysi]³⁺for a 72 hour incubation, and labeled with MTT, according to embodimentsof the present invention.

FIG. 39B is a graph comparing the percent viability of HCT116N (solid)and HCT116O (lined) cells treated with (+=2 mM, or ++=3 mM) or withoutthe PARP inhibitor 3-AB, and with or without 20 μM [Rh(HDPA)₂chrysi]³⁺for a 72 hour incubation, and labeled with MTT, according to embodimentsof the present invention.

FIG. 39C is a graph comparing the percent viability of HCT116N (solid)and HCT116O (lined) cells treated with (+=10 μM, or ++=10 μM) or withoutthe PARP inhibitor 4-AN, and with or without 20 μM [Rh(HDPA)₂chrysi]³⁺for a 72 hour incubation, and labeled with MTT, according to embodimentsof the present invention.

FIG. 39D is a graph comparing the percent viability of HCT116N (solid)and HCT116O (lined) cells treated with (+=5 μM, or ++=10 μM) or withoutthe PARP inhibitor ABT-888, and with or without 20 μM[Rh(HDPA)₂chrysi]³⁺ for a 72 hour incubation, and labeled with MTT,according to embodiments of the present invention.

FIG. 40 is a schematic showing a cellular response to metalloinsertorcomplexes, according to embodiments of the present invention.

FIG. 41 is a schematic showing cell death in response to incorporationof a metalloinsertor complex, according to embodiments of the presentinvention.

FIG. 42 is a schematic showing subcellular localization in response tothe hydrophilic or lipophilic character of the ancillary ligand of ametalloinsertor complex, according to embodiments of the presentinvention.

DETAILED DESCRIPTION

Rhodium complexes bearing the sterically expansive 5,6-chrysene diimine(chrysi) ligand are known to bind selectively to mismatched sites induplex DNA in vitro. The x-ray crystal structures of Rh(bpy)₂chrysi³⁺bound to mismatches show a novel insertion binding mode in which thechrysi ligand displaces the mismatched bases from the minor groove.These chrysi complexes of rhodium are capable of selectively inhibitingthe cellular proliferation of cells deficient in their mismatch repair(MMR) machinery. Improvements to the design of mismatch specific rhodiumcomplexes include reducing potential steric interactions using smallerancillary ligands, improving the mismatch binding affinity throughmodification of the inserting ligand, and optimizing the scaffold forcellular uptake and conjugate development.

It is to be understood that unless otherwise indicated this invention isnot limited to specific reactants, reaction conditions, ligands, metalcomplexes, or the like, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting. Forexample, the present invention is directed to both rhodium and rutheniumcomplexes. However, a person having ordinary skill in the art wouldrecognize that the Rh metal in the exemplified complexes may besubstituted with Ru, and would be capable of making that substitutionbased on the methodology disclosed here.

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

The terms “compounds of Formula I” and “compounds of the invention” and“metalloinsertor complexes of the present invention” are usedinterchangeably and all of these terms refer to a complex represented byFormula I as described above and throughout.

Also, as used herein, unless otherwise indicated, the term “rhodiumcomplex” refers to a complex of Formula I, and is not limited tocomplexes of Formula I in which M is Rh, but rather also includescomplexes of Formula I in which M is Ru.

The term “chrysi” refers to bidentate ligand 5,6-chrysene quinonediimine.

The term “phzi” refers to benzo[a]phenazine-5,6-quinonediimine. The term“phzi complex” or “phzi” to denote a complex, refers to a rhodium (orruthenium) complex having a phzi ligand.

The term “DPAE” refers to 2-(di(pyridin-2-yl)amino)ethanol. The term“DPAE complex” or “DPAE” to denote a complex, refers to a rhodium (orruthenium) complex having a DPAE ligand.

The term “HDPA” refers to 2,2′dipyridylamine. The term “HDPA complex” or“HDPA” to denote a complex, refers to a rhodium (or ruthenium) complexhaving a HDPA ligand.

The term “MeDPA” refers to N-methyl-N-(pyridin-2-yl)pyridin-2-amine. Theterm “MeDPA complex” or “MeDPA” to denote a complex, refers to a rhodium(or ruthenium) complex having a MeDPA ligand.

The term “EtDPA” refers to N-ethyl-N-(pyridin-2-yl)pyridin-2-amine. Theterm “EtDPA complex” or “EtDPA” to denote a complex, refers to a rhodium(or ruthenium) complex having a EtDPA ligand.

The terms “PropylDPA” and “PrDPA” refer toN-propyl-N-(pyridin-2-yl)pyridin-2-amine. The terms “PrDPA complex” or“PrDPA” to denote a complex, refers to a rhodium (or ruthenium) complexhaving a PrDPA ligand.

The term “HexylDPA” refers to N-hexyl-N-(pyridin-2-yl)pyridin-2-amine.The term “HexylDPA complex” or “HexylDPA” to denote a complex, refers toa rhodium (or ruthenium) complex having a HexylDPA ligand.

The term “DPK” refers to di(2-pyridyl) ketone. The term “DPK complex” or“DPK” to denote a complex, refers to a rhodium (or ruthenium) complexhaving a DPK ligand.

The term “DPE” refers to 1,1-di(pyridin-2-yl)ethanol. The teen “DPEcomplex” or “DPE” to denote a complex, refers to a rhodium (orruthenium) complex having a DPE ligand.

The term “bpy” refers to 2,2′-bipyridine. The term “bpy complex” or“bpy” to denote a complex, refers to a rhodium (or ruthenium) complexhaving a bpy ligand.

The term “phen” refers to 1,10-phenanthroline. The term “phen complex”or “phen” to denote a complex, refers to a rhodium (or ruthenium)complex having a phen ligand.

The term “DIP” refers to 4,7-diphenyl-1,10-phenanthroline. The term “DIPcomplex” or “DIP” to denote a complex, refers to a rhodium (orruthenium) complex having a DIP ligand.

The term “rac” refers to racemic mixtures of delta and lambdaenantiomers.

Embodiments of the present invention are directed to rhodium (Rh) andruthenium (Ru) metalloinsertor complexes that target DNA mismatch andselectively induce cytotoxicity. In some embodiments of the presentinvention, methods for identifying DNA mismatch include the use of ametalloinsertor complex as described herein. Other methods of thepresent invention include using a metalloinsertor complex as describedherein for selectively inducing cytotoxicity. Still other methods of thepresent invention include using a metalloinsertor complex as describedherein for selectively decreasing cell proliferation.

In some embodiments of the present invention, a metalloinsertor complexthat targets DNA mismatch is represented by Formula I.

M^(m+)(L₁)(L₂)(L₃)(L₄)(L₅)  Formula I

In Formula I, M is rhodium or ruthenium, and m is 2 or 3. L₁ isbenzo[a]phenazine-5,6-diimine s (phzi) or chrysene-5,6-diimine (chrysi),as depicted below.

In Formula I, each of L₂ through L₅ is either NH₃ or combines with anadjacent one of L₂ through L₅ to form a single ligand with twocoordination sites to M, where the single ligand with two coordinationsites to M is selected from:

orIn Formula II and Formula III, R₁ is selected from alkyl groupsterminating in one selected from CH₃, OH, SH, NH₂, COOH, N₃, oralkynyl-linked peptide moieties, or PEGylated groups terminating in oneselected from CH₃, OH, SH, NH₂, COOH, N₃, or alkynyl-linked peptidemoieties.

Non-limiting examples of specific alkyl derivatives of Formula II,include ligands represented by the following Formula II(a).

In Formula II(a), as shown, R₂ is CH₃, OH, SH, NH₂, COOH, N₃, or analkynyl-linked peptide; and n is any number from 1 to 10. Non-limitingexamples of ligands satisfying Formula II(a) include MeDPA, EtDPA,PrDPA, HexDPA, and DPAE.

Non-limiting examples of PEGylated derivatives of Formula II, includeligands represented by the following Formula II(b).

In Formula II(b), as shown, X is O or NH; R₃ is CH₃, OH, SH, NH₂, COOH,N₃, or an alkynyl-linked peptide moiety; and n is any number from 1 to10.

In some embodiments of the present invention, a complex of Formula I inwhich any of L₂ through L₅ is a ligand represented by Formula II(a) orFormula II(b) allows for conjugation of antibodies, carbohydrates orpeptides to thereby increase cellular accumulation, specificity and/orbiological activity. Indeed, in Formula II (and II(a) and II(b)), R1 canbe an alkyl or PEGylated group terminating in an alkynyl-linked peptidemoiety. The alkynyl-linked peptide can be any suitable such moiety, e.g.an antibody or carbohydrate.

In some embodiments of the present invention, at least two of L₂ throughL₅ are NH₃. In these embodiments, the central M atom is coordinated toat least four ligands. Indeed, when two of L₂ through L₅ are NH₃, theremaining two of L₂ through L₅ may combine to form a single ligandhaving two coordination sites to the central M atom. Non-limitingexamples of suitable ligands for the single ligand having twocoordination sites include those described above, e.g. bpy, phen, DPE,HDPA, DPK, and ligands represented by Formula II.

In other embodiments, all four of L₂ through L₅ are NH₃. In theseembodiments, the central M atom is coordinated to five ligands, i.e.,the four NH₃ ligands and the phzi or chrysi ligand of L₁.

In still other embodiments, none of L₂ through L₅ are NH₃, and L₂ and L₃combine to form a first single ligand having two coordination sites tothe M atom, and L₄ and L₅ combine to form a second single ligand havingtwo coordination sites to the M atom. In these embodiments, the centralM atom is coordinated to three ligands, and each ligand has twocoordination sites to the M atom. Also, in these embodiments, the firstand second single ligands having two coordination sites to the M atomsmay be the same or may be different from each other. Non-limitingexamples of suitable ligands the first and second single ligands, havingtwo coordination sites to the M atom include those described above withrespect to Formulae I and II, e.g., bpy, phen, DPE, HDPA, DPK, andligands represented by Formula II. In some embodiments, for example,both the first and second single ligands can be the same, and can beeither HDPA, MeDPA, PrDPA or DPAE. In some alternative examples,however, the first and second single ligands can be different from eachother, and each of the first and second ligands can be independently oneof DPK, DPE, HDPA, MeDPA, EtDPA, PrDPA, phen, HexDPA, or DPAE.

Non-limiting examples of suitable metalloinsertor complexes includeM^(m+)(L₁)(DPE)(NH₃)₂, M^(m+)(L₁)NH₃)₄, M^(m+)(L₁)(HDPA)₂,M^(m+)(L₁)(MeDPA)₂, M^(m+)(L₁)(MDPA)₂(phen), M^(m+)(L₁)(PrDPA)₂(phen),M^(m+)(L₁)(HexDPA)₂(phen), M^(m+)(L₁)(PrDPA)₂, M⁺(L₁)(DPAE)₂,M^(m+)(L₁)(HDPA)(phen), and M^(m+)(L₁)(DPE)(phen). In these examples, Mand m are as described above, i.e., M is either Rh or Ru, and m is 2 or3. Also, L₁ is as described above, i.e., L₁ is either chrysi or phzi.Non-limiting examples of suitable metalloinsertor complexes satisfyingthese formulae include M^(m+)(DPK)(NH₃)₂(chrysi),M^(m+)(DPE)(NH₃)₂(chrysi), M^(m+)(NH₃)₄(phzi), M^(m+)(HDPA)₂(phzi),M^(m+)(MeDPA)₂(phzi), M^(m+)(MeDPA)₂(chrysi),M^(m+)(MeDPA)(phen)(chrysi), M^(m+)(EtDPA)(phen)(chrysi),M^(m+)(PrDPA)(phen)(chrysi), M^(m+)(HexDPA)(phen)(chrysi),M^(m+)(PrDPA)₂(chrysi), M^(m+)(DPAE)₂(chrysi),M^(m+)(HDPA)(phen)(chrysi), and M^(m+)(DPE)(phen)(chrysi). In theseexamples, M is Rh or Ru, and m is 2 or 3.

Non-limiting examples of suitable rhodium metalloinsertor complexes areshown in FIGS. 1A through 1N. As shown in FIGS. 1A through 1N, exemplarymetalloinsertor complexes include using [Rh(DPK)(NH₃)₂chrysi]³⁺,[Rh(DPE)(NH₃)₂chrysi]³⁺, [Rh(NH₃)₄phzi]³⁺, [Rh(HDPA)₂phzi]³⁺,[Rh(MeDPA)₂phzi]³⁺, [Rh(MeDPA)₂chrysi]³⁺, [Rh(chrysi)(phen)(MeDPA)]³⁺,[Rh(chrysi)(phen)(EtDPA)]³⁺, [Rh(chrysi)(phen)(PrDPA)]³⁺,[Rh(phen)(hexylDPA)chrysi]³⁺, [Rh(PrDPA)₂(chrysi)]³⁺,[Rh(DPAE)₂(chrysi)]³⁺, [Rh(chrysi)(phen)(HDPA)]³⁺ and[Rh(chrysi)(phen)(DPE)]³⁺. Synthesis of each complex is described below.General syntheses for [Rh(DPAE)₂chrysi]³⁺ and [Rh(PrDPA)₂chrysi]³⁺ areshown in FIGS. 28 and 29.

The metalloinsertor complexes of Formula I as described herein,accelerate cellular uptake compared to other metalloinsertor complexes,and therefore trigger a selective cytotoxic effect as a function ofmismatch repair (MMR) status. Accordingly, the metalloinsertor complexesare not only useful for indicating the presence of polynucleotide damageor error, and diagnosing conditions characterized by polynucleotidedamage or error, but are also useful for inducing selective cytotoxicityin cells characterized by polynucleotide damage or error such as cancercells.

In some embodiments of present invention, a method of selectivelyinducing cytotoxicity in an MMR-deficient cell includes providing ametalloinsertor complex of Formula I to an MMR-deficient cell. FIGS.40-42 are schematics showing the mechanism of action for cytotoxicityand subcellular localization of metalloinsertor complexes of Formula Iaccording to the present invention.

The metalloinsertor complexes according to embodiments of the presentinvention, selectively decrease cell proliferation. As such, a methodfor selectively decreasing cell proliferation in MMR-deficient cellsincludes providing a metalloinsertor complex of Formula I to theMMR-deficient cells. This method of selectively decreasing cellproliferation may be in vitro or in vivo. For example, decreasing cellproliferation in vitro includes growing an MMR-deficient cell in thepresence of a metalloinsertor complex of Formula I. Cell cultureconditions suitable for the selected MMR-deficient cell line aredisclosed in the art and below. For another example, decreasing cellproliferation in vivo includes providing or administering ametalloinsertor complex of Formula I to an animal or human.

In other embodiments of the present invention, metalloinsertor complexesof Formula I selectively induce cytotoxicity in MMR-deficient cells. Amethod for selectively inducing cytotoxicity includes providing ametalloinsertor complex of Formula I to MMR-deficient cells. This methodof selectively inducing cytotoxicity may be in vitro or in vivo. Forexample, selectively inducing cytotoxicity in vitro includes growing anMMR-deficient cell in the presence of a metalloinsertor complex ofFormula I. In another example, selectively inducing cytotoxicity in vivoincludes providing or administering a metalloinsertor complex of FormulaI to an animal or human.

In some embodiments of the present invention, a complex of Formula I maybe administered orally or parenterally, for example by injection,inhalation, transdermally, and the like, and may be administered in vivoor ex vivo. For example, one can use compounds of the invention to purgebone marrow of tumor cells prior to reintroducing the marrow into apatient (e.g., after radiotherapy). The compounds can be administeredsystemically or locally, for example via indwelling catheter,controlled- or sustained-release implant, minipump, and the like.Alternatively, the compounds can be formulated as an aerosol, andadministered to the lungs and trachea.

The compounds can be formulated in a liquid dosage form such as, forexample, liquids, suspensions or the like, preferably in unit dosageforms suitable for single administration of precise dosages. Liquiddosages may be administered by injection or infusion, as nose drops oras an aerosol. Alternatively, the active compound can be prepared as acream or an ointment composition and applied topically. As anotheralternative, delivery may occur by controlled release of these agents byencapsulation either in bulk or at a microscopic level using syntheticpolymers, such as silicone, and natural polymers such as gelatin andcellulose. The release rate can be controlled by proper choice of thepolymeric system used to control the diffusion rate (Langer, R. S. andPeppas, N. A., Biomaterials (1981) 2:201). Natural polymers, such asgelatin and cellulose slowly dissolve in a matter of minutes to hourswhile silicone remains intact for a period of months. The compositionswill include a conventional pharmaceutical carrier or excipient, inaddition to one or more of the active compound(s). In addition, thecompositions may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, etc.

The amount of compound administered will of course, be dependent on thesubject being treated, the severity of the affliction, the manner ofadministration, the frequency of administration, and the judgment of theprescribing physician. Suitable concentrations to determine the“effective amount” can be determined by one of ordinary skill in theart, using only routine experimentation. The frequency of administrationis desirably in the range of an hourly dose to a monthly dose,preferably from 8 times/day to once every other day, more preferably 1to 3 times per day. Ointments containing one or more active compoundsand optional pharmaceutical adjuvants in a carrier, such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, can be prepared using a base such as, for example, petroleumjelly, lard, or lanolin.

Liquified pharmaceutically administrable compositions can, for example,be prepared by dissolving, dispersing, etc. an active compound asdefined above and optional pharmaceutical adjuvants in a carrier, suchas, for example, water, saline, aqueous dextrose, glycerol, ethanol, andthe like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. The preparation can additionally contain compounds thatfacilitate entry of the nucleic acid of interest into the inner earcells such as Lipofectin, permeability-enhancing agents (e.g.,detergents), or other transformation-enhancing agents. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 15th Ed., 1975. Thecomposition or formulation to be administered will, in any event,contain a quantity of one or more of the active compound(s) in an amounteffective to alleviate the symptoms of the subject being treated.

For aerosol administration, the active ingredient is preferably suppliedin finely divided form along with a surfactant and a propellant. Typicalpercentages of active ingredients are 0.001 to 2% by weight, preferably0.004 to 0.10%.

Surfactants must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, eleostearic andoleic acids with an aliphatic polyhydric alcohol or its cyclic anhydridesuch as, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, and hexitol anhydrides derived from sorbitol (thesorbitan esters sold under the trademark “Spans”) and thepolyoxyethylene and polyoxypropylene derivatives of these esters. Mixedesters, such as mixed or natural glycerides, may be employed. Thepreferred surface-active agents are the oleates orbita, e.g., those soldunder the trademarks “Arlacel C” (sorbitan sesquioleate), “Span 80”(sorbitan monoleate) and “Span 85” (sorbitan trioleate). The surfactantmay constitute 0.1=20% by weight of the composition, preferably 0.25-5%.

The balance of the composition is ordinarily propellant. Liquefiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane;fluorinated or fluorochlorinated alkanes, such as are sold under thetrademark “Freon”. Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

The following Examples are presented for illustrative purposes only, anddo not limit the scope or content of the present application.

EXAMPLES

The entire contents of Ernst et al., 2011, Biochemistry, 50, 10919-10928are incorporated herein by reference. In the below examples, thefollowing materials are used, unless otherwise indicated.

RhCl₃ was purchased from Pressure Chemical, Inc. (Pittsburgh, Pa.).[Rh(NH₃)₅Cl]Cl₂ was obtained from Strem Chemical, Inc. (Newburyport,Mass.). 2,2′-dipyridylamine (HDPA) and Sephadex ion exchange resin wereobtained from Sigma-Aldrich (St. Louis, Mo.). Sep-Pak C₁₈ solid phaseextraction cartridges were purchased from Waters Chemical Co. (Milford,Mass.). Media and supplements were purchased from Invitrogen (Carlsbad,Calif.). The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) labeling reagent and acidified lysis buffer (10% SDS in 10mM HCl) were purchased in kit format from Roche Molecular Biochemicals(Mannheim, Germany). Z-VAD-FMK caspase inhibitor was purchased fromPromega. All PARP inhibitors were purchased from Santa CruzBiotechnology, Inc. All commercial materials were used as received.

EXAMPLE 1 Synthesis of Tert-butyl 2-(di(pyridine-2-yl)amino)acetate(DPA)

Tert-butyl 2-(di(pyridine-2-yl)amino)acetate was prepared withmodification to Kirin et al., 2007, J. Inorg. Chem., 3686-3694, theentire contents of which are incorporated herein by reference. Potassiumhydroxide (3.0 g, 53.6 mmol, 4.6 equiv) was added to a solution of2,2′-dipyridylamine (2.0 g, 11.7 mmol) in 40 ml DMSO and stirred at roomtemperature for 16 hours (h). Potassium iodide (200 mg, 1.2 mmol, 0.1equiv) and tent-butyl bromoacetate (4 ml, 2.3 equiv) were added to themixture, and the reaction was stirred for 2 h at room temperature. Thereaction mixture was extracted with diethyl ether (3×50 ml). The organicfractions were combined and dried over magnesium sulfate, and thesolvent was removed by rotary evaporation. The crude product wasisolated by flash chromatography (SiO₂, hexane/ethyl acetate=8:2) togive a yellow oil. DPA was obtained (Yield: 2.92 g (88%)), and confirmedby ¹H NMR (CDCl₃, 300 MHz): δ8.33 (ddd, J=5.0, 1.9, 0.9 Hz; 2H), 7.53(m, 2H), 7.23 (m, 2H), 6.88 (ddd, J=7.2, 5.0, 0.9 Hz; 2H), 4.84 (s, 2H),1.42 (s, 9H). ESI-MS (cation): 286 m/z (M+H⁺) obsd, 286 m/z calcd.

EXAMPLE 2 Synthesis of 2-(di(pyridine-2-yl)amino)ethanol

To a slurry of LAH (lithium aluminum hydride) (1.17 g, 30.8 mmol, 3.0equiv) in THF (tetrahydrafuran) (45 ml) was added Tert-butyl2-(di(pyridine-2-yl)amino)acetate (2.9 g, 10.2 mmol) at 0° C. under 1atm argon (Ar). The reaction was slowly warmed to room temperature over4 h. The reaction mixture was then diluted with diethyl ether and cooledto 0° C. The reaction was quenched via careful addition of water (4.0ml) and then dried with magnesium sulfate. The solvent was removed invacuo, and the crude product was purified by flash chromatography (SiO₂,hexane/ethyl acetate=1:1) to afford DPAE as a pale yellow oil. DPAE wasobtained (Yield: 1.2 g (55%)), and confirmed by ¹H NMR (DMSO-d₆, 300MHz): δ8.27 (m, 2H), 7.62 (m, 2H), 7.16 (d, J=8.4 Hz, 2H), 6.93 (m, 2H),4.92 (t, J=5.4 Hz, 1H), 4.16 (t, J=6.5 Hz, 2H), 3.58 (q, J=6.5 Hz, 2H).ESI-MS (cation): 216.1 m/z (M+H⁺) obsd, 215 m/z calcd.

EXAMPLE 3 Synthesis of 1,1-di-2-pyridylethanol

DPE was prepared according to Basu et al., 1987, Journal of the Chem.Soc, Chemical Comm., 22, 1724-1725, the entire contents of which areincorporated herein by reference.

EXAMPLE 4 Synthesis of N-alkyl-N-(pyridin-2-yl)pyridin-2-amine (MeDPA),EtDPA, PropylDPA, HexylDPA)

To a slurry of sodium hydride (70 mg, 2.9 mmol) in THF (10 ml) was addedHDPA (500 mg, 2.9 mmol) in 5 ml THF at 0° C. under 1 atm Ar. Thereaction was purged with argon for 15 min, and the appropriate1-bromomethane (3.8 mmol) was added dropwise and warmed to roomtemperature. The reaction was stirred an additional 18 h under argon atreflux temperature. The reaction mixture was extracted with dilutesodium bicarbonate, and the aqueous phase was extracted with CH₂Cl₂(3×40 ml). The organic fractions were combined and dried over magnesiumsulfate, and the solvent was removed in vacuo. MethylDPA was obtained(10 mmol scale, Yield: 0.44 g, 23%), and confirmed by ¹H-NMR (CDCl₃):8.35 (d of d, 2H); 7.54 (t, 2H); 7.17 (d, 2H); 6.86 (t, 2H), 3.62 (s,3H). ESI-MS: 186 m/z [M+H]⁺.

EXAMPLE 5 Synthesis of N-ethyl-N-(pyridin-2-yl)pyridin-2-amine (EtDPA)

EtDPA was synthesized as described in Example 4, except that1-bromoethane was used instead of 1-bromomethane. EthylDPA was obtained(1 mmol scale, Yield: 65.5 mg, 32%), and confirmed by ¹H-NMR (CDCl₃):δ8.39-8.26 (m, 2H), 7.56-7.40 (m, 2H), 7.06 (dd, J=8.4, 0.4 Hz, 2H),6.87-6.76 (m, 2H), 4.23 (q, J=7.0 Hz, 2H), 1.30-1.12 (m, 5H).

EXAMPLE 6 Synthesis of N-propyl-N-(pyridin-2-yl)pyridin-2-amine (PrDPA).

PrDPA was synthesized as described in Example 4, except that1-bromopropane was used instead of 1-bromomethane. PropylDPA wasobtained (10 mmol scale, Yield: 0.76 g, 36%), and confirmed by ¹H NMR(300 MHz, cdcl₃) δ8.39-8.20 (m, 2H), 7.59-7.40 (m, 2H), 7.05 (dd, J=8.4,0.8 Hz, 2H), 6.81 (ddd, J=6.7, 5.4, 0.8 Hz, 2H), 4.16-4.01 (m, 2H),1.78-1.59 (m, 2H), 0.91 (t, J=7.4 Hz, 3H).

EXAMPLE 7 Synthesis of N-hexyl-N-(pyridin-2-Apyridin-2-amine (HexylDPA)

HexylDPA was synthesized as described in Example 4, except that1-bromohexane was used instead of 1-bromomethane. HexylDPA was obtained(10 mmol scale, Yield: 0.46 g, 18%), and confirmed by ¹H NMR (300 MHz,cdcl₃) δ8.35-8.25 (m, 2H), 7.55-7.41 (m, 2H), 7.05 (d, J=8.4 Hz, 2H),6.81 (dd, J=7.1, 5.0 Hz, 2H), 4.23-4.03 (m, 2H), 1.63 (m, 2H), 1.25 (m,6H), 0.83 (t,3H).

EXAMPLE 8 Synthesis of [Rh(DPK)(NH₃)₂chrysi]³⁺

A 100-mL round-bottomed flask was charged with [Rh(NH₃)₄chrysi]Cl₃ (15.0mg, 27.7 μmol) and DPK (7.7 mg, 41.6 μmol), as described in Muerner etal., 1998, Inorg. Chem. 37, 3007-3012, the entire contents of which areincorporated herein by reference. Ethanol (30 mL) was added and theresulting red solution was stirred at 60° C. for 16 hours. The solventwas evaporated in vacuo and the resulting red solid purified via columnchromatography (C₁₈-derivatized silica, eluting with 10% acetonitrile in0.1% TFA_((aq))). The fractions containing product were identified byHPLC, combined, and lyophilized. The chloride salt can be obtained froma Sephadex QAE anion exchange column equilibrated with 0.1M MgCl₂.[Rh(NH₃)₄chrysi]Cl₃ was obtained (Yield: 9 mg, 35%). and confirmed byESI-MS: calc. 575.1 (M−2H⁺), obs. 574.8 (M−2H⁺), 288.6 (M−H²⁺).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium complex, [Ru(DPK)(NH₃)₂chrysi]⁺, can be made bythe same process by substituting the [Rh(NH₃)₄chrysi]Cl₃ with[Ru(NH₃)₄chrysi]Cl₂.

EXAMPLE 9 Synthesis of [Rh(chrysi)(DPE)(NH₃)₂]Cl₃

A 250 mL round-bottomed flask was charged with [Rh(chrysi)(NH₃)₄]TFA₃(25 mg, 0.059 mmol) and 1,1-di(pyridin-2-yl)ethanol (36.5 mg, 0.182mmol) in ethanol (50 mL) to give a red solution. The reaction was heatedto reflux (80° C.) and stirred for 5 days. The ethanol solution wasevaporated to dryness and dissolved in 0.1% TFA_((aq)) (100 mL). The redsolution was loaded onto a solid phase extraction (SPE) cartridge andrinsed with copious amount of 0.1% TFA_((aq)). The SPE cartridge waseluted with 10% acetonitrile in 0.1% TFA_((aq)) and fractions werecollected. The fractions containing product were identified by HPLC,combined, and lyophilized. The chloride salt can be obtained from aSephadex QAE anion exchange column equilibrated with 0.1M MgCl₂.[Rh(chrysi)(DPE)(NH₃)₂]Cl₃ was obtained (Yield: 18 mg, 43%), andconfirmed by ESI-MS: calc. 591.49 (M−2H⁺), obs. 591.1 (M−2H⁺).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound, [Ru(chrysi)(DPE)(NH₃)₂]Cl₂, can bemade by the same process by substituting the [Rh(chrysi)(NH₃)₄]TFA₃ with[Ru(chrysi)(NH₃)₄]TFA₂.

EXAMPLE 10 Synthesis of [Rh(MeDPA)₂chrysi]Cl₃

[Rh(NH₃)₄chrysi]TFA₃ (15 mg, 0.02 mmol) was reacted with MeDPA (22 mg,0.12 mmol) in a 1:1 mixture of ethanol: water (40 mL). The dark redsolution was heated under reflux (95° C.) for 16 hr. Ethanol was removedunder vacuum, and the resulting reddish brown solution was filtered toremove any residue. The filtrate was loaded onto a SPE cartridge andeluted with 25% acetonitrile in 0.1% TFA_((aq)). The resulting reddishsolid was further purified via column chromatography (C₁₈-derivatizedsilica, eluting with 12.5% acetonitrile in 0.1% TFA_((aq))). Thefractions containing product were identified by HPLC, combined, andlyophilized to give a red solid. The chloride salt can be obtained froma Sephadex QAE anion exchange column equilibrated with 0.1M MgCl₂.[Rh(MeDPA)₂chrysi]Cl₃ was obtained (Yield: 7.5 mg, 35%), and confirmedby ESI-MS: calc. 727.2 (M−2H⁺), obs. 727.1 (M−2H⁺), 364.3 (M−H^(2±)).UV-Vis (H₂O, pH 7): 295 nm (55,000 M⁻¹ cm⁻¹), 320 nm (39,700 M⁻¹, cm⁻¹),390 nm (14,000 M⁻¹, cm⁻¹).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound, [Ru(MeDPA)₂chrysi]Cl₂, can be made bythe same process by substituting the [Rh(NH₃)₄chrysi]TFA₃ with[Ru(NH₃)₄chrysi]TFA₂.

EXAMPLE 11 Synthesis of [Rh(NH₃)₄phzi]Cl₃

[Rh(NH₃)₆][OTf]₃ (0.50 g, 0.77 mmol) and benzo[a]phenazine-5,6-dione(0.200 g, 0.77 mmol) were dissolved in a 1:5 mixture of water:acetonitrile (500 mL). A 1M solution of NaOH (1 mL) was added to theyellow solution and the reaction was allowed to stir at room temperaturefor 45 min, at which time a 1M solution of HCl (1 mL) was added toneutralize the reaction mixture. The acetonitrile was evaporated invacuo and the resulting yellow solution was loaded onto a SPE cartridge,eluted with 25% acetonitrile in 0.1% TFA_((aq)), and lyophilized to givea yellow solid. The chloride salt can be obtained from a Sephadex QAEanion exchange column equilibrated with 0.1M MgCl₂. [Rh(NH₃)₄phzi]Cl₃was obtained (Yield: 0.45 g, 76%), and confirmed by ¹H NMR (300 MHz,d₆-DMSO): δ3.79 (s, 6H), 4.48 (d, J=20.1 Hz, 6 H), 7.92-8.21 (m, 4H),8.34 (m, 2H), 8.62 (d, J=7.6 Hz, 1H), 8.96 (d, J=7.8 Hz, 1 H), 13.88 (s,1H), 13.98 (s, 1H). ESI-MS: calc. 524.07 (M−NH₃+TFA⁺)., obs. 523.8(M−NH₃+TFA⁺). UV-Vis (H₂O, pH 7): 250 nm (36,800 M⁻¹ cm⁻¹), 310 nm(20,800 M⁻¹, cm⁻¹), 340 nm (23,400 M⁻¹, cm⁻¹).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound, [Ru(NH₃)₄phzi]Cl₂, can be made by thesame process by substituting the [Rh(NH₃)₆][OTf]₃ with [Ru(NH₃)₆][OTf]₂.

EXAMPLE 12 Synthesis of [Rh(HDPA)₂phzi]Cl₃

A solution of [Rh(NH₃)₄phzi]TFA₃ (53.2 mg, 69.2 μmol in ethanol (25 mL)was added to a 100-mL schlenk flask and sparged with argon for 10 min. Asolution of HDPA (0.346 mmol) in ethanol (25 mL) was then added and theresulting light yellow solution sparged with argon for an additional 10min, then subsequently heated to 90° C. The solution was allowed to stirfor 48 hours, after which the solvent was evaporated in vacuo and theresulting red solid purified via column chromatography (C₁₈-derivatizedsilica, eluting with 12.5% acetonitrile in 0.1% TFA_((aq))). Thefractions containing product were identified by HPLC, combined, andlyophilized to give a red solid. The chloride salt can be obtained froma Sephadex QAE anion exchange column equilibrated with 0.1M MgCl₂.[Rh(HDPA)₂phzi]Cl₃ was obtained (Yield: 21.5 mg, 38%), and confirmed by¹H NMR (300 MHz, d₆-DMSO): δ7.04 (m, 4H), 7.81 (m, 5H), 7.92 (m, 5H),8.03 (m, 4H), 8.14 (m, 3H), 8.27 (m, 3H), 8.73 (s, 1H), 9.37 (s, 1H),12.65 (s, 1H), 13.02 (s, 1H). ESI-MS: calc. 701.16 (M−2H⁺)., obs. 701.1(M−2H⁺), 351.3 (M−H²⁺). UV-Vis (H₂O, pH 7): 318 nm (44,600 M⁻¹ cm⁻¹),350 nm (33,200 M⁻¹, cm⁻¹), 400 nm (9,100 M⁻¹, cm⁻¹).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compounds, [Ru(HDPA)₂phzi]Cl₂ can be made by thesame process by substituting the [Rh(NH₃)₄phzi]TFA₃ with[Ru(NH₃)₄phzi]TFA₂.

EXAMPLE 13 Synthesis of [Rh(MeDPA)₂phzi]Cl₃

[Rh(MeDPA)₂phzi]Cl₃ was synthesized as described in Example 12, exceptthat MeDPA was substituted for HDPA. [Rh(MeDPA)₂phzi]Cl₃ Yield: 18.7 mg,32%. ¹H NMR (300 MHz, d₆-DMSO): δ3.87 (s, 3H), 4.06 (s, 3H), 7.24 (m,5H), 7.67 (m, 3H), 7.73 (m, 3H), 7.97 (m, 5H), 8.11 (m, 4H), 8.28 (m,4H), 14.37 (s, 1H), 15.16 (s, 1H). ESI-MS: calc. 729.19 (M−2H⁺), obs.729.2 (M−2H⁺), 544.1 (M−L−2H⁺), 365.4 (M−H²⁺). UV-Vis (H₂O, pH 7): 306nm (45,000 M⁻¹ cm⁻¹), 340 nm (32,500 M⁻¹, cm⁻¹), 400 nm (10,700 M⁻¹,cm⁻¹).

EXAMPLE 14 Synthesis of [Rh(chrysi)(phen)(NH₃)₂]Cl₃

was prepared from [Rh(phen)(NH₃)₄]OTf₃ and 5,6-chrysenequinone followingthe methods described by Muerner et. al, 1998 (supra).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound, [Ru(chrysi)(phen)(NH₃)₂]Cl₂, can bemade by the same process by substituting the [Rh(phen)(NH₃)₄]OTf₃ with[Ru(phen)(NH₃)₄]OTf₂.

EXAMPLE 15 Synthesis of [Rh(chrysi)(phen)(HDPA)]Cl₃

[Rh(chrysi)(phen)(NH₃)₂]Cl₃ (25 mg, 0.02 mmol) was reacted with HDPA(0.022 mmol) in a 4:1 mixture of ethanol:water (10 mL). The bright redsolution was refluxed overnight. The solvent was removed in vacuo andthe product was filtered to remove any residue. The filtrate wasconcentrated on a SPE cartridge, eluted with 20% acetonitrile inTFA_((aq)), and lyophilized to give a red solid. Analytically purematerial was obtained from reverse phase HPLC and lyophilized to yield adark red solid. The chloride salt can be obtained from a Sephadex QAEanion exchange column equilibrated with 0.1 M MgCl₂.[Rh(chrysi)(phen)(HDPA)]Cl₃ was obtained (Yield: 28%), and confirmed byESI-MS: calc 708.14 [M−2H⁺], obs. 708.2 (M−2H+) UV-Vis (H₂O, pH 7): 303nm (57000 M⁻¹ cm⁻¹), 391 nm (10,600 M⁻¹, cm⁻¹).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound, [Ru(chrysi)(phen)(HDPA)]Cl₂, can bemade by the same process by substituting the Rh(chrysi)(phen)(NH₃)₂]Cl₃with Ru(chrysi)(phen)(NH₃)₂]Cl₂.

EXAMPLE 16 Synthesis of [Rh(chrysi)(phen)(MeDPA)]Cl₃

[Rh(chrysi)(phen)(MeDPA)]Cl₃ was synthesized as described in Example 15,except that MeDPA was substituted for HDPA. [Rh(chrysi)(phen)(MeDPA)]Cl₃was obtained (Yield: 32%), and confirmed by ¹H NMR (500 MHz, DMSO-d₆)δ12.45 (s, 1H), 10.48 (s, 1H), 9.34-6.83 (m, 26H), 3.87 (s, 3H). ESI-MS:calc. 722.15 (M−2H⁺), obs. 722 (M−2H⁺), 362 (M−H²⁺).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound [Ru(chrysi)(phen)(MeDPA)]Cl₂ can bemade by the same process by substituting the Rh(chrysi)(phen)(NH₃)₂]Cl₃with Ru(chrysi)(phen)(NH₃)₂]Cl₂.

EXAMPLE 17 Synthesis of [Rh(chrysi)(phen)(EtDPA)]

[Rh(chrysi)(phen)(EtDPA)] was synthesized as described in Example 15,except that EtDPA was substituted for HDPA. [Rh(chrysi)(phen)(EtDPA)]Cl₃was obtained, (Yield: 28%), and confirmed by ESI-MS: calc. 736.17(M−2H⁺), obs. 736 (M−2H⁺), 369 (M−H²⁺).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound [Ru(chrysi)(phen)(EtDPA)]Cl₂ can bemade by the same process by substituting the Rh(chrysi)(phen)(NH₃)₂]Cl₃with Ru(chrysi)(phen)(NH₃)₂]Cl₂.

EXAMPLE 18 Synthesis of [Rh(chrysi)(phen)(PropylDPA)]Cl₃

[Rh(chrysi)(phen)(PropylDPA)]Cl₃ was synthesized as described in Example15, except that PrDPA was substituted for HDPA.[Rh(chrysi)(phen)(PrDPA)]Cl₃ was obtained (Yield: 22%), and confirmed byESI-MS: calc. 750.18 (M−2H⁺), obs. 750 (M−2H⁺), 376 (M−H²⁺).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound [Ru(chrysi)(phen)(PrDPA)]Cl₂ can bemade by the same process by substituting the Rh(chrysi)(phen)(NH₃)₂]Cl₃with Ru(chrysi)(phen)(NH₃)₂]Cl₂.

EXAMPLE 19 Synthesis of [Rh(chrysi)(phen)(HexylDPA)] Cl₃

[Rh(chrysi)(phen)(HexylDPA)]Cl₃ was synthesized as described in Example15, except that hexylDPA was substituted for HDPA.[Rh(chrysi)(phen)(HexylDPA)]Cl₃ was obtained (Yield: 18%), and confirmedby ESI-MS: calc. 792.23 (M−2H⁺), obs. 792 (M−2H⁺), 397 (M−H²⁺).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound [Ru(chrysi)(phen)(HexylDPA)]Cl₂ can bemade by the same process by substituting the Rh(chrysi)(phen)(NH₃)₂]Cl₃with Ru(chrysi)(phen)(NH₃)₂]Cl₂.

EXAMPLE 20 Synthesis of [Rh(chrysi)(phen)(DPE)] Cl₃

A 125 mL round-bottomed flask was charged with[Rh(chrysi)(phen)(NH₃)₂]TFA₃ (62.0 mg, 0.068 mmol) and DPE (25.6 mg,0.128 mmol) in ethanol (50 mL) to give a red solution. The reaction washeated to reflux (80° C.) and stirred for 48 hours. The ethanol solutionwas evaporated to dryness and dissolved in 0.1% TFA_((aq))(trifluoroacetic acid) (50 mL). The red solution was loaded onto a SPEcartridge and rinsed with copious amount of 0.1% TFA_((aq)). The SPEcartridge was eluted with 10% acetonitrile in 0.1% TFA_((aq)) andfractions were collected. The fractions containing product wereidentified by HPLC, combined, and lyophilized. The chloride salt can beobtained from a Sephadex QAE anion exchange column equilibrated with0.1M MgCl₂. [Rh(chrysi)(phen)(DPE)]Cl₃ was obtained (Yield: 17 mg,29.6%), and confirmed by ESI-MS: calc. 737.15 (M−2H⁺), obs. 737 (M−2H⁺),369 (M−H²⁺). UV-Vis (H₂O, pH 7): 272 nm (59800 M⁻¹ cm⁻¹), 303 nm (18,400M⁻¹, cm⁻¹), 440 nm (5,400 M⁻¹, cm⁻¹).

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium compound, [Ru(chrysi)(phen)(DPE)]Cl₂, can bemade by the same process by substituting the[Rh(chrysi)(phen)(NH₃)₂]TFA₃ with [Ru(chrysi)(phen)(NH₃)₂]TFA₂.

EXAMPLE 21 Synthesis of [Rh(DPAE)₂chrysi]³⁺

[Rh(NH₃)₄chrysi]Cl₃ (20 mg, 0.038 mmol) and DPAE (17.8 mg, 0.082 mmol,excess) were dissolved in a 1:1 mixture of ethanol and water (100 ml)and heated under reflux for 28 h. The solvent was removed in vacuo, andthe crude product was purified by HPLC (95:5:0.001water:acetonitrile:TFA), using a C18 reverse-phase column (Varian, Inc).The purified product was dried under vacuum and redissolved in a minimalvolume of water. The TFA counterion was exchanged for a chloride with aSephadex QAE-125 ion-exchange resin primed with 1M MgCl₂.[Rh(chrysi)(phen)(DPE)]Cl₃ was obtained (Yield: 4.5 mg (13.5%)), andconfirmed by ¹H NMR (300 MHz, DMSO-d6) δ13.03 (s, 1H), 9.27 (d, J=8.1Hz, 1H), 9.02-8.75 (m, 5H), 8.60-8.49 (m, 2H), 8.45-7.60 (m, 31H), 7.52(d, J=5.8 Hz, 1H), 7.48-7.31 (m, 3H), 7.31-7.09 (m, 4H), 5.34 (s, 2H),4.97 (s, 3H), 4.67 (s, 2H), 4.48 (s, 3H), 4.32 (s, 1H), 4.11 (s, 1H),3.82 (s, 2H), 3.61 (s, 5H). UV-vis (H₂O pH 8): 297 nm (47,000 M⁻¹ cm⁻¹),391 nm (9,300 M⁻¹ cm⁻¹). ESI-MS (cation): 787.1 m/z (M−2H⁺), 394.2 m/z(M−H²⁺) obsd, 787 m/z (M−2H⁺) calcd.

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium complex, [Ru(DPAE)₂chrysi]²⁺, can be made by thesame process by substituting the [Rh(NH₃)₄chrysi]Cl₃ with[Ru(NH₃)₄chrysi]Cl₂.

EXAMPLE 22 Synthesis of [Rh(PrDPA)₂chrysi]³⁺

[Rh(PrDPA)₂chrysi]³⁺ was synthesized from [Rh(NH₃)₄chrysi]Cl₃ (20 mg,0.038 mmol) and PropylDPA (17 mg, 0.08 mmol) as described for[Rh(DPAE)₂chrysi]³⁺. The resulting product was purified by HPLC95:5:0.001 H₂O:acetonitrile:TFA) and passed through a Sephadex QAE-125ion-exchanged resin primed with 1M MgCl₂ to give the chloride salt.[Rh(PrDPA)₂chrysi]³⁺ was obtained (Yield: 3 mg (15%).), and confirmed by¹H NMR (300 MHz, DMSO-d6) δ10.10 (s, 2H), 8.11 (s, 2H), 8.00 (d, J=9.1Hz, 4H), 7.76 (s, 3H), 7.62 (d, J=8.5 Hz, 13H), 7.36 (d, J=6.6 Hz, 9H),7.24 (s, 4H), 6.83 (d, J=7.9 Hz, 9H), 1.72 (s, 7H), 1.49 (s, 2H), 1.10(s, 7H), 0.97-0.84 (m, 6H), 0.62 (t, J=7.2 Hz, 9H), 0.31 (s, 1H), 0.00(t, J=7.3 Hz, 9H). UV-vis: (H₂O pH 8): 295 nm (51,000 M⁻¹ cm⁻¹), 388 nm(13,000 M⁻¹ cm⁻¹). ESI-MS (cation): 783.1 m/z (M−2H⁺), 392.4 m/z (M−H²⁺)obsd, 783 m/z calcd.

As would be understood by those of ordinary skill in the art, thecorresponding ruthenium complex, [Ru(PrDPA)₂chrysi]²⁺, can be made bythe same process by substituting the [Rh(NH₃)₄chrysi]Cl₃ with[Ru(NH₃)₄chrysi]Cl₂.

EXAMPLE 23 Synthesis of Ruthenium (Ru) Complexes

As would be understood by those having ordinary skill in the art, thesynthesis scheme shown below can be easily adapted to make Ru analogs ofany of the Rh complexes as disclosed herein by substitution of theappropriate ligands. The synthesis scheme shown is adapted from Brunneret al., 2006, Biochemistry, 45, 12295 and Copeland et al., 2002,Biochemistry, 41, 12785, the entire contents of both of which areincorporated herein by reference.

EXAMPLE 24 PEGylation

Methods for PEGylation for drug delivery are known in the art. Forexample, Greenwald et al., 2003, Adv. Drug Del. Rev., 55, 217; Molineux,2003, Pharmacotherapy, (8 Pt 2), 3S-8S; Roberts et al., 2002, Adv. DrugDeliv. Rev., 54, 459; Greenwald, 2001, J. Controlled Release 74, 159,and Veronese et al., 1995, Adv. Drug Deliv. Rev. 76, 157, the entirecontents of all of which are incorporated herein by reference.

Methodology for Metalloinsertor Complex Assays

The metalloinsertor complexes as synthesized and described were assayedin cellulo using the isogenic colorectal carcinoma cell lines HCT116Nand HCT116O. The methods used are as follows.

EXAMPLE 25 Cell Culture

HCT116N and HCT116O cells were grown in RPMI medium 1640 supplementedwith: 10% FBS; 2 mM L-glutamine; 0.1 mM nonessential amino acids; 1 mMsodium pyruvate; 100 units/mL penicillin; 100 μg/mL streptomycin; and400 μg/mL geneticin (G418). Cells were grown in tissue culture flasksand dishes (Corning Costar, Acton, Mass.) at 37° C. under 5% CO₂ andhumidified atmosphere.

EXAMPLE 26 Cellular Proliferation ELISA

The ability of each of the complexes of FIGS. 1A through 1N toselectively inhibit cell growth was assayed using a BrdU(5-bromo-2′-deoxyrudine) incorporation assay in the colorectal carcinomacell lines HCT116N and HCT116O. BrdU is a synthetic thymidine analogthat is incorporated into newly synthesized DNA during the S-phase ofthe cell cycle in growing cells. The HCT116N and HCT116O cell linesdiffer only in the presence of an active copy of the human MLH1 gene,which is essential for mismatch repair (MMR). HCT116N contains theactive MLH1 gene, and is MMR proficient, while HCT116O lacks an activeMLH1 gene and is MMR-deficient.

The results of the BrdU assays for each of these complexes in both theHCT116N and HCT116O cell lines are shown in FIGS. 2-15B. FIG. 16 is agraph showing the percent of selective inhibition of cell growth usingmetalloinsertor complexes of the present invention as indicated. HCT116Nand HCT116O cells were plated in 96-well plates at 2,000 cells/well andallowed 24 hours to adhere. The cells were then incubated with rhodiumfor the durations specified. For incubation less than 72 hours, theRh-containing media was replaced with fresh media, and the cells weregrown for the remainder of the 72 hour period. Cells were labeled withBrdU 24 hours before analysis. The BrdU incorporation was quantified byantibody assay as described in Reitmar et al., 1997, Cancer Res, 57,3765-3771 and Gratzner 1982, Science, 218, 474-475, the entire contentsof both of which are incorporated herein by reference. Cellularproliferation was expressed as the ratio of the amount of BrdUincorporated by the treated cells to that of the untreated cells.

EXAMPLE 27 MTT Cytotoxicity Assay

Cytotoxicity assays were performed as described in Mosmann, 1983, J.Immunol. Methods, 65, 55-63, the entire contents of which isincorporated herein by reference. HCT116N and HCT116O cells were platedin 96-well plates at 50,000 cells/well and incubated with rhodium forthe durations specified. After incubation with the indicated rhodiumcomplexes, cells were labeled with MTT for 4 hours at 37° C. under 5%CO₂ and humidified atmosphere. The resulting formazan crystals weredissolved with solubilizing reagant purchased from Roche according tothe manufacturer's instructions. The dissolved formazan was quantifiedas the absorbance at 570 nm minus the background absorbance at 690 nm.Percent viability was determined as the ratio of the amount of formazanin the treated cells to that of the untreated cells.

The results from cytotoxicity assays using [Rh(DPAE)₂chrysi]³⁺ in theHCT116N and HCT116O cells are shown in FIGS. 17A-17C. As shown, the DPAEcomplex exhibits cell selective activity as early as 6 hours. That is,the DPAE complex imparts increased cytotoxicity to the MMR-deficientHCT116O cells. FIGS. 18A-18C show the results from cytotoxicity assaysusing [Rh(PrDPA)₂chrysi]³⁺ in HCT116N and HCT116O cells. As shown, thePrDPA complex imparts non-selective inhibitory effects after 72 hours.

FIGS. 19A and 19B show the cytotoxicity results using the knownnon-toxic [Rh(bpy)₂chrysi]³⁺ complex in the HCT116N and HCT116O cells.FIGS. 20A and 21B show cytotoxicity results using [Rh(HDPA)₂chrysi]³⁺,and FIGS. 21A and 21B show the cytotoxicity results using[Rh(MeDPA)₂chrysi]³⁺. As shown, both [Rh(HDPA)₂chrysi]³⁺[Rh(MeDPA)₂chrysi]³⁺ complexes enhance toxicity in the MMR-deficientHCT116O cells at 48 hours. At 48 hours [Rh(HDPA)₂chrysi]³⁺ and[Rh(MeDPA)9chrysi]³⁺ clearly display an enhanced toxicity in theMMR-deficient HCT116O cell line versus the HCT116N cell line (FIGS. 20A,20B, 21A, and 21B). For example, 72 hours after treatment with 20 μM[Rh(HDPA)₂chrysi]³⁺, the number of viable HCT116N cells is 80±5.2% ofuntreated controls, whereas the number of viable HCT116O cells is37±4.4% of untreated controls. [Rh(MeDPA)₂chrysi]³⁺ also showsdifferential toxicity against the HCT116O cell line comparable to thatof [Rh(HDPA)₂chrysi]³⁺.

FIGS. 22-27 show the results from cytotoxicity assays using[Rh(NH3)₄phzi]³⁺, [Rh(chrysi)(phen)(DPE)]³, [Rh(chrysi)(phen)(HDPA)]³[Rh(chrysi)(phen)(MeDPA)]³, [Rh(chrysi)(phen)(PrDPA)]³, and[Rh(DIP)₂(chrysi)]³ in HCT116N and HCT116O cells, respectively. Asshown, [Rh(NH3)₄phzi]³⁺ and [Rh(chrysi)(phen)(DPE)]³ exhibit selectivecytotoxicities towards the MMR-deficient HCT116O cells.

EXAMPLE 28 Binding Competition Titrations

For FIGS. 30A-30C, a 29-mer DNA hairpin containing a CC mismatch(underlined) (*{tilde over (5)}GGCAGGCATG-GCTTTTTGCCATCCCTGCC-3′) waslabeled with ³²P at the 5′-end as described in Zeglis et al., 2007, Nat.Protoc. 2, 357-371, the entire contents of which are incorporated hereinby reference. A 1:1 mixture of labeled and unlabeled DNA was prepared inbuffer (100 mM NaCl, 20 mM NaP_(i), pH 7.1) to a final concentration of2 μM. The hairpin was annealed by heating to 90° C. for 10 min andslowly cooled to room temperature. To prepare samples for gelelectrophoresis, 4 μM [Rh(bpy)₂chrysi]Cl₃ (5 μL) and varyingconcentrations of rac-Rh(DPAE)₂chrysi³⁺ or rac-Rh(PrDPA)₂chrysi³ (5 μL)were added to 2 μM annealed DNA hairpin (10 μL). A light control (10 μLDNA, 10 μL H₂O), a dark control (10 μL DNA, 5 μL Rh(bpy)₂chrysi³⁺, 5μLrac-Rh(DPAE)₂chrysi³⁺ or rac-Rh(PrDPA)₂chrysi³, no irradiation), and apositive control (10 μL DNA, 5 μL Rh(bpy)₂chrysi³⁺, 5 μL H₂O) were alsoprepared. Samples were vortexed and, except for the dark control,irradiated on an Oriel (Darmstadt, Germany) 1000-W Hg/Xe solar simulator(340-440 nm) for 15 min. Samples were then incubated at 37° C. for 20min, dried, then electrophoresed through a 20% denaturing polyacrylamidegel. The gel was exposed on a phosphor screen, phosphorimaged, and theamounts of DNA cleavage were quantified using ImageQuant.

To determine the K_(B) values of rac-Rh(DPAE)₂chrysi³⁺ andrac-Rh(PrDPA)₂chrysi³⁺ competition gels were run in triplicate for eachcomplex, and the percent DNA cleavage at each concentration was averagedand plotted as a function of log [Rh] (FIG. 30C). The data were fit to asigmoidal curve using OriginPro 8.1. K_(B) values were determined bycalculating the concentration of rhodium at the inflection points of thecurve and solving simultaneous equilibria involving DNA,Rh(bpy)₂chrysi³⁺ and rac-Rh(DPAE)₂chrysi³⁺ or rac-Rh(PrDPA)₂chrysi³inMathematica 8.0. The dissociation constant K_(D) is defined as 1/K_(B).

For the autoradiograms of rac-Rh(DPAE)₂chrysi³⁺ (FIG. 30A) andrac-Rh(PrDPA)₂chrysi³⁺ (FIG. 30B) a light control (Light, withoutrhodium), dark control (Dark, without irradiation), and positive control(Positive, only Rh(bpy)₂chrysi³⁺) are included. Concentrations ofcompetitors Rh(DPAE)₂chrysi³⁺ and Rh(PrDPA)₂chrysi³⁺ range from 0.1-50μM, with 1 μM Rh(bpy)₂chrysi³⁺.

EXAMPLE 29 ICP-MS Assay for Cellular Rhodium Levels

To assay the subcellular localization of the rhodium complexes, aninductively coupled plasma mass spectrometry (ICP-MS) assay was used toquantify the uptake of the complexes in both whole cell extracts andnuclear and mitochondrial fractions.

Each cell line was treated with 10 μM of each rhodium complex asindicated in FIG. 33A (except [Rh(DIP)9(chrysi]³⁺ which was administeredat 2 μM) for 1, 3, 6, 12, 24, or 48 hours. After rhodium incubation, thecells were harvested from adherent culture by trypsinization, washedwith cold PBS, and counted by hemacytometer. The samples were pelletedand resuspended in 1% HNO₃ (v/v), homogenized by three freeze/thawcycles in liquid nitrogen, and analyzed for rhodium content on anHP-4500 ICP-MS unit. Rhodium counts were normalized to the number ofcells counted in each sample before lysate preparation. Standard errorsfor three independent experiments are shown.

Whole-Cell Rhodium Accumulation. With reference to FIG. 31A, HCT116Ocells were plated in 6-well plates at 1.0×10⁶ cells/well (3 ml media),and allowed 24 h to adhere. The cells were then incubated with 10 μMrac-Rh(DPAE)₂chrysi³⁺ or rac-Rh(PrDPA)₂chrysi³ for an additional 24 h.Cells were lysed with 1% SDS and sonicated. Samples were aliquoted (0.75ml) and diluted with 2% HNO₃ (0.75 ml), and cellular rhodium content wasquantified on an HP-4500 ICP-MS unit. The remainder of the cell lysateswere analyzed for protein content via bicinchoninic acid (BCA) assay.Rhodium counts were normalized to protein content, and standard errorswere calculated from three replicates.

Mitochondrial Rhodium Accumulation. With reference to FIG. 31B, HCT116Ocells were plated in 75 cm² culture flasks at 2.0×10⁷ cells/plate andincubated at 37° C., 5% CO₂ for 24 h. Rhodium was added to 10 uM andcells were grown for an additional 24 h. The cells were then harvestedby trypsinization and centrifuged for 5 min at 1,200 rpm. Thesupernatants were decanted, and the cell pellets were resuspended in 1ml cold PBS (pH 7.2). The cells were centrifuged again for 5 min at1,200 rpm. The supernatants were discarded, and the resultant pelletswere resuspended in 0.5 ml mitochondrial extraction buffer (200 mMmannitol, 68 mM sucrose, 50 mM Pipes, 50 mM KCl, 5 mM EGTA, 2 mM MgCl₂;1 mM DTT and protease inhibitors were added right before use). Thesamples were incubated on ice for 20 min, and the suspensions werehomogenized via passage through a needle and syringe (35×). Thehomogenized cells were then centrifuged for 5 min at 750 rpm. Thesupernatants were collected and spun again at 14,000 g for 10 min. Thesupernatants were decanted, and the resulting mitochondrial pellet wassuspended in 0.8 ml H₂O via probe sonication. All samples were diluted1× with 2% HNO₃. 20 uL aliquots were used in a BCA assay to determineprotein content, which was carried out according to standard protocol.Rh counts from ICP MS were converted to ppb and normalized to proteincontent (ng Rh/mg protein). The purity of mitochondrial fractions wasascertained by Western blot.

Nuclear Rhodium Accumulation. With reference to FIG. 31B, HCT116O cellswere plated in 75 cm² culture flasks at 1.5×10⁷ cells/plate andincubated at 37° C., 5% CO₂, for 24 h. Rhodium was then added to 10 μMand cells were grown for an additional 24 h. The cells were trypsinizedaccording to standard protocol, and the cell pellets were washed with 3mL 1×PBS (pH 7.2) and spun at 1200 rpm for 5 minutes. The supernatantwas discarded, and the pellets were resuspended in 1 mL 1×PBS anddivided into 2×0.5 mL aliquots (nuclear and whole cell). The sampleswere spun at 450 g for 5 minutes at 4° C. The supernatants were decantedand the whole cell pellets were dissolved in 1 mL Milli-Q water. Thenuclear pellets were dissolved in 1 mL hypotonic buffer (20 mM Tris-HCl,pH 7.4; 10 mM NaCl, 3 mM MgCl₂) and incubated on ice for 15 min. After15 min, 50 uL of NP-40 detergent were added and the samples werevortexed for 10 s. Samples were then spun at 3000 g for 10 min at 4° C.The supernatants were discarded, and the nuclear pellets were dissolvedin 1 mL Milli-Q water via sonication. All samples were diluted 1× with2% HNO₃. 20 uL aliquots were used in a BCA assay to determine proteincontent, which was carried out according to standard protocol. Rh countsfrom ICP MS were converted to ppb and normalized to protein content (ngRh/mg protein).

As shown in FIGS. 31A and 31B the mitochondrial rhodium content in cellsincubated with [Rh(PrDPA)₂(chrysi)]³⁺ is greater than that of cellsincubated with [Rh(DPAE)₂(chrysi)]³⁺. This difference in localizationcorrelates with the lipophilic characteristics of [Rh(PrDPA)₂(chrysi)]³which facilitate uptake in response to mitochondrial membrane potential.A greater percentage of cellular [Rh(DPAE)9(chrysi)]³⁺ is nuclear.Accordingly, the MMR-selective effects to override any nonspecificconsequences of mitochondrial accumulation.

As shown in FIG. 32, cells incubated with [Rh(HDPA)₂chrysi]³⁺ exhibit anincrease in cellular uptake compared to cells incubated with[Rh(bpy)₂chrysi]³ or [Rh(NH₃)₄chrysi]³. FIGS. 33A and 33B show theamount of cellular uptake and localization for the indicated rhodiumcomplexes.

EXAMPLE 30 Cell Cycle Distribution Flow Cytometry Assay

Cells were harvested from adherent culture by trypsinization and washedwith cold PBS. The resultant pellet was resuspended in PBS (chilled to4° C.), and ice-cold ethanol was added dropwise to a final concentrationof 70% (v/v), with continuous gentle agitation. Cells were fixed at 4°C. for 30 minutes and stored for up to one week. Prior to analysis, thefixed cells in 70% ethanol were diluted 1:3 in cold PBS and centrifugedat 1,400×g for 5 minutes. The resultant pellet was washed twice andresuspended in ice-cold PBS. Ribonuclease was added to a finalconcentration of 30 μg/mL and the cells were incubated overnight at 4°C. The next day propidium iodide was added to a final concentration of20 μg/mL and cells were analyzed by flow cytometry. Data analysis wasperformed using the FloJo software package (v 8.7.1).

EXAMPLE 31 Cell Death Mode Flow Cytometry Assay

To characterize the cell death occurring in response to rhodiumtreatment, a dye exclusion flow cytometry assay was used as described inIdziorek et al., 1995 J. Immunol. Methods, 185, 249-258, the entirecontents of which is incorporated herein by reference. The assaydifferentiates between live cells, dead cells, and cells undergoingapoptosis or necrosis through concurrent staining with propidium iodide(a dead cell permeable dye) and YO-PRO-1 (an apoptotic cell permeabledye). By plotting the fluorescence of the YO-PRO-1 channel against thePI channel, a pattern emerges. Healthy cells are seen in the lowerlefthand corner of the plot in FIGS. 36A-36D. Apoptotic cells exhibithigher YO-PRO-1 fluorescence, but still exclude propidium iodide,placing them in the upper lefthand quadrant of the pattern. Dead cellsadmit both dyes and are therefore seen in the upper righthand quadrantof the image. Upon flow cytometry analysis, cells can be classified aslive, apoptotic, necrotic, or dead by defining regions in thefluorescence plane corresponding to each category.

The HCT116N and HCT116O cell lines were incubated with 0-25 μM of[Rh(HDPA)₂chrysi]³⁺ for 24-72 hours. After harvesting the cells andstaining with both PI and YO-PRO-1, the cells were analyzed by flowcytometry to obtain raw fluorescence data. Representative data for 20 μMrhodium treatment for 72 hours are shown. YO-PRO-1 fluorescence is shownon the y-axis, and PI fluorescence is shown on the x-axis. The raw datawere analyzed by gating the fluorescence events into one of fourcategories, depending on the fluorescence levels of the two dyes. FIGS.37A-37B also show histograms of live, apoptotic, necrotic, and deadcells for the HCT116N and HCT116O cell lines based on the flowcytometry. Treatment with the indicated rhodium complex was either 15 or20 μM [Rh(HDPA)₂chrysi]³⁺ for 72 hours. As before, rhodium treatmentalone induces necrosis preferentially in the MMR-deficient HCT116O cellline; there is no significant change in the percentage of cells in theapoptotic region in either cell line. The effect is significantly morepronounced in the MMR-deficient HCT116O cell line, which drops from79±3.8% to 37±5.3% after treatment with 20 μM [Rh(HDPA)₂chrysi]³⁺,versus the MMR-proficient HCT116N cell line, which shows a minimaldecrease in live cells from 62±0.6% to 54±5.1% after treatment with 20μM [Rh(HDPA)₂chrysi]³⁺.

After 24, 48, or 72 hour incubation with the selected rhodium complex,cells were harvested from adherent culture by trypsinization and washedwith cold PBS, and centrifuged at 2,000 rpm for 5 minutes. The resultantpellets were resuspended in PBS to a concentration of 10⁶ cells/mL andstained with propidium iodide to a final concentration of 1 μg/mL andwith YO-PRO-1 (an apoptotic cell permeable dye) to a final concentrationof 200 nM for 30 minutes prior to analysis by flow cytometry.

EXAMPLE 32 Caspase Inhibition

In order to elucidate the mechanism of action for the cytotoxicitycaused by a metalloinsertor complex of Formula I, the MTT cytotoxicityassay was repeated in the absence and presence of the pan-caspaseinhibitor Z-VAD-FMK. This inhibitor works by irreversibly binding to theactive site of caspases which participate in the apoptotic pathway.Z-VAD-FMK was added to a final concentration of 20 μM. The HCT116N andHCT116O cell lines were treated with 0-30 μM of the [Rh(HDPA)₂chrysi]³⁺complex for 24-72 hours. In addition, each treatment was also combinedwith the inhibitor at a final concentration of 20 μM. The rhodiumcomplex exhibited selective toxicity in the repair-deficient HCT116Ocell line, with cell viability dropping to 9.7±4.4% after treatment with30 μM metal complex for 72 hours, versus 63±15.7% viability in therepair-proficient HCT116N cell line. Addition of the caspase inhibitorat 20 μM offered no protection from rhodium to the HCT116N cell line(63±5.7% without inhibitor, 52±9.8% with inhibitor) or to the HCT116Ocell line (9.7±4.4% without inhibitor, 9.8±7.8% with inhibitor). At afinal concentration of 40 μM, the caspase inhibitor provided someprotection from rhodium to the HCT116O cell line (16±10% withoutinhibitor, 28±3.7% with inhibitor), but this difference was small inrelation to the differential between the HCT116N and HCT116O cell linesand roughly within error.

FIGS. 34A-34D, FIGS. 35A-35B, FIGS. 36A-36D, and FIGS. 37A-37B showresults from flow cytometry assays of [Rh(HDPA)₂chrysi]3⁺ HCT116N andHCT116O cells. As shown, selective cytotoxicity (cell death) is precededby disruption of the cell cycle, and that cell death proceeds through anecrotic rather than apoptotic pathway. The caspase inhibition assayusing shown in FIGS. 38A-38C shows that the selective cell death iscaspase-independent—i.e. not apoptotic.

EXAMPLE 33 PARP Inhibition

In order to determine the mechanism of action for cytotoxicity caused bya metalloinsertor complex of Formula I, the MTT cytotoxicity assay wasrepeated in conjunction with a panel of poly-ADP ribose polymerase(PARP) inhibitors: DPQ, 3-AB, 4-AN, and ABT-888, as described inCostantino et al., 2001, J. Med. Chem., 44, 3786-3794; Purnell et al.,1980, Biochem. J. 185, 775-777; Banasik et al., 1992, J. Biol. Chem.267, 1569-1575; and Donawho et al., 2007, Clin. Cancer Res., 13,2728-2737, the entire contents of all of which are incorporated hereinby reference. The HCT116N and HCT116O cell lines were treated with 0 or20 μM of the [Rh(HDPA)₂chrysi]³⁺ complex for 72 hours, with or withoutone of the four inhibitors. The inhibitor3,4-Dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline (DPQ) wasadded to a final concentration of 0, 25, or 50 μM; the inhibitor3-aminobenzamide (3-AB) was added to a final concentration of 0, 2, or 3mM; the inhibitor 4-amino-1,8-napthalimide (4-AN) was added to a finalconcentration of 0, 10, or 20 μM; and the inhibitor2-((R)-2-Methylpyrrolidin-2-yl)-1H-benzimidazole-4-carboxamide (ABT-888)was added to a final concentration of 0, 5, or 10 μM. In each case,treatment with the inhibitor completely abolished the selectiveMMR-dependent effects of the rhodium compound, as determined by thedifference between the percentage of viable cells in the HCT116N cellline and the percentage of viable cells in the HCT116O cell line. Forexample, in the case of the compound DPQ, this difference was 43±2.7%without inhibitor and 0.6±3.0% with inhibitor. Similar results are seenwith each of the other three compounds as well; taken together, thesedata, as shown in FIGS. 39A-39D, indicate that PARP participates in theMMR-dependent response to [Rh(HDPA)₂chrysi]³⁺.

As disclosed throughout and evidenced by the selective cytotoxicity dataof FIGS. 17A through 27, metalloinsertor complexes of the presentinvention provide a means for selectively targeting MMR-deficient cells.

While the present invention has been illustrated and described withreference to certain exemplary embodiments, those of ordinary skill inthe art will understand that various modifications and changes may bemade to the described embodiments without departing from the spirit andscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A composition comprising a complex represented byFormula I:M^(m+)(L₁)(L₂)(L₃)(L₄)(L₅)  Formula I wherein M is rhodium or ruthenium;m is 2 or 3; L₁ is benzo[a]phenazine-5,6-diimine orchrysene-5,6-diimine;

wherein each of L₂ through L₅ is either NH₃ or combines with an adjacentone of L₂ through L₅ to form a single ligand with two coordination sitesto M, where the single ligand with two coordination sites to M isselected from the group consisting of:

and and wherein, in Formula II and Formula III, R₁ is selected from thegroup consisting of: alkyl groups terminating in one selected from thegroup consisting of CH₃, OH, SH, NH₂, COOH, N₃, and alkynyl-linkedpeptide moieties, and PEGylated groups terminating in one selected fromthe group consisting of CH₃, OH, SH, NH₂, COOH, N₃, and alkynyl-linkedpeptide moieties.
 2. The composition of claim 1, wherein at least two ofL₂ through L₅ combine to form the single ligand having two coordinationsites to M, and the single ligand having two coordination sites to M isrepresented by Formula II(a):

wherein R₂ is selected from the group consisting of CH₃, OH, SH, NH₂,COOH, N₃, and alkynyl-linked peptide moieties; and n is any number from1 to
 10. 3. The composition of claim 1, wherein at least two of L₂through L₅ combine to form the single ligand having two coordinationsites to M, and the single ligand having two coordination sites to M isrepresented by Formula II(b):

wherein X is O or NH; R₃ is selected from the group consisting of CH₃,OH, SH, NH₂, COOH, N₃, and alkynyl-linked peptide moieties; and n is anynumber from 1 to
 10. 4. The composition of claim 1, wherein at least twoof L₂ through L₅ combine to form the single ligand having twocoordination sites to M, and the single ligand having two coordinationsites to M comprises a ligand selected from the group consisting of DPE,HDPA, MeDPA, MDPA, phen, PrDPA, HexDPA, and DPAE.
 5. The composition ofclaim 1, wherein L₂ and L₃ combine to form a first single ligand havingtwo coordination sites to M, L₄ and L₅ combine to form a second singleligand having two coordination sites to M, and each of the first andsecond single ligands having two coordination sites to M independentlycomprises a ligand selected from the group consisting of DPE, HDPA,MeDPA, MDPA, phen, PrDPA, HexDPA, and DPAE.
 6. The composition of claim1, wherein M is rhodium.
 7. The composition of claim 1, wherein at leasttwo of L₂ through L₅ combine to form the single ligand having twocoordination sites to M, and the single ligand having two coordinationsites to M is represented by Formula II in which R₁ is selected from thegroup consisting of: alkyl groups terminating in an alkynyl-linkedpeptide moiety comprising an antibody or a carbohydrate, and PEGylatedgroups terminating in an alkynyl-linked peptide moiety comprising anantibody or a carbohydrate.
 8. The composition of claim 1, wherein atleast two of L₂ through L₅ comprise NH₃.
 9. The composition of claim 8,wherein at least two of L₂ through L₅ combine to form the single ligandhaving two coordination sites to M, and the single ligand having twocoordination sites to M is selected from the group consisting of DPE,HDPA, MeDPA, MDPA, phen, PrDPA, HexDPA, and DPAE.
 10. The composition ofclaim 1, wherein all of L₂ through L₅ comprise NH₃.
 11. The compositionof claim 1, wherein the complex represented by Formula I comprises acomplex selected from the group consisting of M^(m+)(L₁)(DPE)(NH₃)₂,M^(m+)(L₁)(NH₃)₄, M^(m+)(L₁)(HDPA)₂, M^(m+)(L₁)(MeDPA)₂,M^(m+)(L₁)(MDPA)₂(phen), M^(m+)(L₁)(PrDPA)₂(phen),M^(m+)(L₁)(HexDPA)₂(phen), M^(m+)(L₁)(PrDPA)₂, M^(m+)(L₁)(DPAE)₂,M^(m+)(L₁)(HDPA)(phen), and M^(m+)(L₁)(DPE)(phen).
 12. The compositionof claim 1, wherein the complex represented by Formula I comprises acomplex selected from the group consisting of M^(m+)(DPK)(NH₃)₂(chrysi),M^(m+)(DPE)(NH₃)₂(chrysi), M^(m+)(NH₃)₄(phzi), M^(m+)(HDPA)₂(phzi),M^(m+)(MeDPA)₂(phzi), M^(m+)(MeDPA)₂(chrysi),M^(m+)(MeDPA)(phen)(chrysi), M^(m+)(EtDPA)(phen)(chrysi),M^(m+)(PrDPA)(phen)(chrysi), M^(m+)(HexDPA)(phen)(chrysi),M^(m+)(PrDPA)₂(chrysi), M^(m+)(DPAE)₂(chrysi),M^(m+)(HDPA)(phen)(chrysi), and M^(m+)(DPE)(phen)(chrysi).
 13. Thecomposition of claim 1, wherein the complex represented by Formula Icomprises a complex selected from the group consisting ofRh³⁺(DPK)(NH₃)₂(chrysi), Rh³⁺(DPE)(NH₃)₂(chrysi), Rh³⁺(NH₃)₄(phzi),Rh³⁺(HDPA)₂(phzi), Rh³⁺(MeDPA)₂(phzi), Rh³⁺(MeDPA)₂(chrysi),Rh³⁺(MeDPA)(phen)(chrysi), Rh³⁺(EtDPA)(phen)(chrysi),Rh³⁺(PrDPA)(phen)(chrysi), Rh³⁺(HexDPA)(phen)(chrysi),Rh³⁺(PrDPA)₂(chrysi), Rh³⁺(DPAE)₂(chrysi), Rh³⁺(HDPA)(phen)(chrysi), andRh³⁺(DPE)(phen)(chrysi).
 14. A method of selectively inducingcytotoxicity in mismatch repair (MMR)-deficient cells, comprising:providing the composition of claim 1 to the MMR-deficient cells.
 15. Themethod of claim 14, wherein providing the composition of claim 1comprises providing the composition in vitro.
 16. The method of claim14, wherein providing the composition of claim 1 comprises providing thecomposition in vivo.
 17. A method of selectively decreasing cellproliferation, comprising: providing the composition of claim 1 toMMR-deficient cells.
 18. A pharmaceutical composition, comprising: aneffective amount of the composition of claim 1; and a pharmaceuticallyacceptable carrier.